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The Journal of Immunology, 2002, 169: 4153-4160.
Copyright © 2002 by The American Association of Immunologists

Enforced Expression of Bcl-2 Restores the Number of NK Cells, But Does Not Rescue the Impaired Development of NKT Cells or Intraepithelial Lymphocytes, in IL-2/IL-15 Receptor {beta}-Chain-Deficient Mice1

Masahiro Minagawa*, Hisami Watanabe{dagger}, Chikako Miyaji{dagger}, Katsuhiro Tomiyama*, Hideki Shimura*, Akiko Ito*, Masaaki Ito*, Jos Domen2,{ddagger}, Irving L. Weissman{ddagger} and Kazuhiro Kawai3,*

Departments of * Dermatology and {dagger} Immunology, Niigata University School of Medicine, Niigata, Japan; and {ddagger} Departments of Pathology and Developmental Biology, Stanford University School of Medicine, Stanford, CA 94305


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
IL-2/IL-15R{beta}-deficient mice display impaired development of NK cells, NKT cells, and intraepithelial lymphocytes of the intestine and skin. To determine the role of survival signals mediated by IL-2/IL-15R in the development of these innate lymphocytes, we introduced a bcl-2 transgene into IL-2/IL-15R{beta}-deficient mice. Enforced expression of Bcl-2 restored the number of NK cells in IL-2/IL-15R{beta}-deficient mice, but the rescued NK cells showed no cytotoxic activity. The numbers of NKT cells and intestinal intraepithelial lymphocytes did not increase significantly, and skin intraepithelial lymphocytes remained undetectable in the bcl-2 transgenic IL-2/IL-15R{beta}-deficient mice. These results indicate an essential role of IL-2/IL-15R-mediated survival signals in the development of NK cells, but they also show that additional nonsurvival signals from IL-2/IL-15R are necessary for innate lymphocyte development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mutations in the common cytokine receptor {gamma}-chain ({gamma}c),4 which is a shared subunit of the receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11), result in X-linked SCID in humans (12). Similarly, mice with targeted mutations in the {gamma}c gene exhibit impaired development of multiple lymphocyte lineages characterized by reduced numbers of conventional {alpha}{beta} T cells and B cells and absence of {gamma}{delta} T cells, NK cells, and intraepithelial lymphocytes (IEL) of the intestine and skin (13, 14, 15, 16). Analyses of mice deficient for an individual {gamma}c-dependent cytokine or cytokine receptor subunit revealed IL-7/IL-7R as crucial for the development of several major lymphocyte lineages. Thus, mice deficient for IL-7 or specific {alpha}-chain of IL-7R (IL-7-/- and IL-7R{alpha}-/- mice) have reduced numbers of conventional {alpha}{beta} T cells and B cells and no {gamma}{delta} T cells (17, 18, 19, 20, 21, 22). However, NK cells and TCR{alpha}{beta} intestinal IEL develop almost normally in these mice (18, 21, 22, 23). In contrast, mice deficient for the shared {beta}-chain of IL-2R and IL-15R (IL-2/IL-15R{beta}-/- mice) display normal development of conventional {alpha}{beta} T cells and B cells but have dramatically reduced numbers of NK cells, TCR{gamma}{delta} and TCR{alpha}{beta}CD8{alpha}{alpha} subsets of intestinal IEL, and NK1.1+TCR{alpha}{beta}+ NKT cells, and no skin IEL (23, 24, 25, 26, 27). Because IFN regulatory factor-1-/- mice (in which the IL-15 gene expression is impaired (28, 29)), IL-15-/- mice, and IL-15R{alpha}-/- mice are similarly deficient in NK cells, NKT cells, and CD8{alpha}{alpha} intestinal IEL (28, 29, 30, 31), and because IL-15-/- mice have no skin IEL (32), the development of these innate lymphocytes may depend primarily on IL-15/IL-15R rather than IL-2/IL-2R. In fact, IL-2-/- mice do not display obvious defects in the development of NK cells, NKT cells, and skin IEL (26, 33, 34), although impaired development of CD8{alpha}{alpha} intestinal IEL in IL-2-/- mice has been demonstrated (23, 34, 35).

Multiple signaling pathways from IL-7R and IL-2/IL-15R have been identified (36), but the signals directly regulating the lymphocyte development are not fully defined. The protein tyrosine kinases Janus kinase (Jak)1, which is associated with IL-7R{alpha} and IL-2/IL-15R{beta} (7, 8, 37), and {gamma}c-associated Jak3 (7, 8, 38) are critical to invoke signals from IL-7R and IL-2/IL-15R (39, 40). Accordingly, both Jak1-/- and Jak3-/- mice display defects in the lymphocyte development similar to those of {gamma}c-deficient mice (39, 41, 42, 43, 44). Essential roles of two distinct downstream signals in the lymphocyte development have been also identified for the IL-7R (45). An important IL-7R-mediated signal is to promote V(D)J recombination in the IgH and TCR{gamma} gene loci in B cell and {gamma}{delta} T cell precursors, respectively (46, 47, 48, 49). Although a role of IL-7R-mediated signals in TCR{beta} locus rearrangements has been also suggested (50), the thymic development of conventional {alpha}{beta} T cell precursors is blocked before TCR{beta} rearrangement in IL-7-/-, IL-7R{alpha}-/-, {gamma}c-deficient, and Jak3-/- mice (17, 20, 23, 44, 51). The thymic precursors in these mice exhibit reduced expression of the antiapoptotic protein Bcl-2 (44, 52, 53, 54), and enforced expression of Bcl-2 by introducing a bcl-2 transgene restores the development of conventional {alpha}{beta} T cells, but not B cells or {gamma}{delta} T cells in IL-7R{alpha}-/- and {gamma}c-deficient mice (51, 53, 55, 56, 57, 58). Therefore, the primary role of IL-7R in the conventional {alpha}{beta} T cell development is to provide survival signals in their thymic precursors independently of the signals promoting VDJ recombination of the TCR{beta} locus.

Comparably little is known about the downstream signals from the IL-2/IL-15R that regulate innate lymphocyte development. Unlike IL-7, IL-2/IL-15 does not activate signals promoting V(D)J recombination in lymphocyte precursors. However, multiple signaling pathways from the IL-2/IL-15R lead to the expression of antiapoptotic proteins Bcl-2 and Bcl-xL in various cell lines as well as in primary T cells (59, 60, 61, 62, 63, 64, 65). In addition, IL-2/IL-15 has been shown to promote survival of mature human NK cells (66, 67) and mouse intestinal TCR{gamma}{delta} IEL (68) through up-regulation of Bcl-2 expression. Therefore, the impaired development of innate lymphocytes in the mice with abrogated IL-2/IL-15R-mediated signals might result from the lack of survival signals.

To determine the role of survival signals mediated by IL-2/IL-15R in the innate lymphocyte development, we have introduced a bcl-2 transgene into IL-2/IL-15R{beta}-/- mice. We show that enforced expression of Bcl-2 in IL-2/IL-15R{beta}-/- mice has differential effects on the development of each innate lymphocyte lineage.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

IL-2/IL-15R{beta}-/- mice and H2K-bcl-2 transgenic mice were reported previously (24, 69) and were maintained on a C57BL/6 background. The bcl-2-IL-2/IL-15R{beta}-/- mice were generated by crossing IL-2/IL-15R{beta}-/- mice with H2K-bcl-2 transgenic mice. C.B-17 SCID mice were purchased from Japan Clea (Tokyo, Japan). All mice were used at 4–5 wk of age.

Cell preparation

Liver lymphocytes, intestinal IEL, and epidermal cells were prepared as described previously (27, 70, 71).

Flow cytometry

Cells were resuspended in PBS supplemented with 2% FCS, 0.1% NaN3, and 25 mM EDTA. After preincubation with anti-Fc{gamma}II/IIIR mAb (clone 2.4G2; BD PharMingen, San Diego, CA), cells were stained with saturating amounts of the following mAbs (BD PharMingen): FITC- or biotin-conjugated anti-TCR{beta} (H57-597), PE- or biotin-conjugated anti-NK1.1 (PK136), PE-conjugated DX5, PE-conjugated anti-CD3-{epsilon} (145-2C11), FITC-conjugated anti-Mac-1 (M1/70), FITC-conjugated anti-Ly-49A (A1), FITC-conjugated anti-Ly-49C/I (5E6), FITC-conjugated anti-Ly-49D (4E5), FITC-conjugated anti-Ly-49G2 (4D11), PE-conjugated anti-TCR{delta} (GL3), FITC-conjugated anti-CD8{alpha} (53-6.7), PE-conjugated anti-CD8{beta} (53-5.8), and PE-conjugated anti-TCR V{gamma}3 (536). Biotin-conjugated mAb was visualized with streptavidin-TRI-color (Caltag Laboratories, Burlingame, CA) or streptavidin-Quantum Red (Sigma-Aldrich, St. Louis, MO). After gating on forward and side scatter and propidium iodide, viable cells were analyzed using the FACScan flow cytometer with the Lysis II or CellQuest program (BD Biosciences, Mountain View, CA). Intracellular staining of transgenic or endogenous Bcl-2, IL-4, or IFN-{gamma} was performed as described (53, 72) using the Cytofix/Cytoperm kit (BD PharMingen) and the following mAbs: FITC-conjugated anti-human Bcl-2 (126; DAKO, Glostrup, Denmark), FITC-conjugated anti-mouse Bcl-2 (3F11), PE-conjugated anti-IL-4 (11B11), PE-conjugated anti-IFN-{gamma} (XMG1.2), and PE-conjugated rat IgG isotype control (R3-34) (all from BD PharMingen).

Cytotoxicity assay

Mice were injected i.p. with 100 µg of poly(I):poly(C) (Pharmacia Biotech, Piscataway, NJ) in 100 µl PBS on days 0 and 1. On day 2, effector cells were prepared and cytotoxic activity against NK-sensitive YAC-1 target cells was determined by the standard 4-h 51Cr release assay (73).

Cytokine production assay

To induce IFN-{gamma} production by NK cells in vitro, spleen cells were cultured (1 x 106 cells/ml) for 4 h in the presence or absence of 1 ng/ml mouse rIL-12 (PeproTech, London, U.K.) and 100 ng/ml mouse rIL-18 (MBL, Nagoya, Japan). GolgiStop (BD PharMingen) containing monensin was added during the last 2 h, and intracellular accumulation of IFN-{gamma} in NK cells was analyzed by flow cytometry.

To stimulate NKT cells in vivo, mice were injected i.p. with 10 µg of {alpha}-galactosylceramide ({alpha}GalCer) (KRN7000; Kirin Brewery, Tokyo, Japan). After 90 min, spleen cells were prepared and cultured (5 x 106 cells/ml) for 1 h. IL-4 levels of the supernatants were determined using an ELISA kit (Genzyme/Techne, Minneapolis, MN). For intracellular staining, cells were incubated in the presence of GolgiStop for 2 h, and intracellular accumulation of IL-4 was analyzed by flow cytometry.

RT-PCR

RNA extraction and reverse transcription using pd(N)6 primers were performed as described (27). Diluted cDNA was amplified by PCR using primers specific for TCR V{alpha}14-J{alpha}281 (74). The amount of template cDNA was normalized using primers for {beta}-actin (27).

Immunofluorescence staining

Five-micrometer frozen sections were cut and fixed in cold acetone. The sections were stained with anti-MHC class II mAb (clone M5/114) or rat IgG isotype control (BD PharMingen) and visualized with FITC-conjugated anti-rat Ig Ab (DAKO).

Statistical analysis

Differences in the absolute numbers of each lymphocyte population were analyzed for statistical significance using the Student t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of bcl-2-IL-2/IL-15R{beta}-/- mice

H2K-bcl-2 transgenic mice, in which the human bcl-2 transgene is driven by the H2Kb promoter and Moloney murine leukemia virus long terminal repeat (69), express the transgenic Bcl-2 in all hematopoietic cells including thymic precursors of conventional {alpha}{beta} T cells, B cell precursors and hematopoietic stem cells in the bone marrow, and spleen NK cells (51, 69). We also confirmed expression of the transgenic Bcl-2 by NK cells and NKT cells in the liver, all intestinal IEL subsets, skin IEL, and their fetal thymic precursors in H2K-bcl-2 transgenic mice (data not shown).

IL-2/IL-15R{beta}-/- mice display impaired development of NK cells, NKT cells, and IEL of the intestine and skin but have normal numbers of conventional {alpha}{beta} T cells and B cells (23, 24, 25, 26, 27). However, in aged IL-2/IL-15R{beta}-/- mice, conventional {alpha}{beta} T cells are spontaneously activated and induce an exhaustive differentiation of B cells into plasma cells, resulting in fatal lymphoproliferative disorders characterized by lymphadenopathy, splenomegaly, high serum concentrations of autoantibodies, anemia, and marked infiltrative granulocytopoiesis (24). These immunopathological phenotypes were not altered in the aged bcl-2-IL-2/IL-15R{beta}-/- mice (data not shown). Therefore, in the following experiments, bcl-2-IL-2/IL-15R{beta}-/- and littermate control mice were used at 4–5 wk of age to minimize possible influences of the lymphoproliferative disorders on the innate immune cell development.

Enforced expression of Bcl-2 restores the number, but not cytotoxic activity, of NK cells in IL-2/IL-15R{beta}-/- mice

Enforced expression of Bcl-2 in IL-2/IL-15R{beta}-/- mice resulted in a significant increase in the number of NK1.1+TCR{beta}- NK cells in the liver and spleen (Figs. 1Go and 2Go), which was comparable to wild-type (+/+) levels. The relative proportion of NK1.1-TCR{beta}+ conventional T cells was decreased by the enforced expression of Bcl-2 (Fig. 1Go), probably due to the increase in the number of B cells. The NK1.1+TCR{beta}- cells in bcl-2-IL-2/IL-15R{beta}-/- mice were CD3-{epsilon}-TCR{delta}-, and Mac-1, DX5, Ly-49A, C/I, D, and G2 were expressed at similar levels by NK cells in the spleens of +/+, IL-2/IL-15R{beta}-/-, and bcl-2-IL-2/IL-15R{beta}-/- mice (Fig. 3Go and data not shown), Therefore, the rescued NK cells in bcl-2-IL-2/IL-15R{beta}-/- mice were phenotypically indistinguishable from mature NK cells in +/+ mice. However, the spleen or liver lymphocytes in bcl-2-IL-2/IL-15R{beta}-/- mice showed no cytotoxic activity against NK-sensitive target cells (Fig. 4Go and data not shown), although the proportion of NK cells in the effector cells of bcl-2-IL-2/IL-15R{beta}-/- mice (6.3 ± 1.1% in the liver lymphocytes) was comparable to that of +/+ mice (5.3 ± 1.7%).



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  		FIGURE 1. Enforced expression of Bcl-2 increases the numbers of NK cells, but not of NKT cells, in IL-2/IL-15R{beta}-/- mice. Liver lymphocytes were stained with anti-TCR{beta} and anti-NK1.1 mAbs. The percentage of each subset is shown. The cell number recovered from each mouse is also shown above the panel. Three mice for each type were analyzed independently and representative profiles are shown.

 


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  		FIGURE 2. Restoration of NK cell number in bcl-2-IL-2/IL-15R{beta}-/- mice. Absolute numbers of NK cells in the liver and spleen of the mice with indicated genotypes were calculated from the total cell number and the percentage of NK cells. The mean and SD of three mice are shown. *, Significant decreases in numbers of NK cells as compared with those in +/+ mice (p < 0.01).

 


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  		FIGURE 3. Similar levels of Mac-1 expression on NK cells in +/+, IL-2/IL-15R{beta}-/-, and bcl-2-IL-2/IL-15R{beta}-/- mice. Spleen cells were stained with anti-CD3-{epsilon}, anti-NK1.1, and anti-Mac-1 mAbs. NK1.1+CD3-{epsilon}- NK cells were gated, and their Mac-1 expression was analyzed. The percentage of Mac-1high and Mac-1low NK cell subpopulations is shown. Two mice for each type were analyzed independently and representative profiles are shown.

 


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  		FIGURE 4. Rescued NK cells in bcl-2-IL-2/IL-15R{beta}-/- mice do not have cytotoxic activity. Liver lymphocytes from poly(I):poly(C)-treated +/+ (•), IL-2/IL-15R{beta}-/- ({blacktriangleup}), and bcl-2-IL-2/IL-15R{beta}-/- ({blacksquare}) mice were used as effector cells against YAC-1 target cells. Representative data from three independent experiments are shown.

 
It is known that NK cells produce large amounts of IFN-{gamma} in response to stimulation with IL-12 and IL-18 (75). Because cytotoxic activity and IFN-{gamma} production are regulated by different pathways in NK cells (76), we next examined the capacity to produce IFN-{gamma} of the rescued NK cells in bcl-2-IL-2/IL-15R{beta}-/- mice. Upon in vitro stimulation with IL-12 and IL-18, spleen NK cells from +/+ and bcl-2-IL-2/IL-15R{beta}-/- mice rapidly produced IFN-{gamma} (Fig. 5Go). Thus, the rescued NK cells in bcl-2-IL-2/IL-15R{beta}-/- mice have no cytotoxic activity but can produce IFN-{gamma}.



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  		FIGURE 5. Rescued NK cells in bcl-2-IL-2/IL-15R{beta}-/- mice can produce IFN-{gamma} in response to in vitro stimulation with IL-12 and IL-18. Spleen cells from +/+ and bcl-2-IL-2/IL-15R{beta}-/- mice were cultured in the presence or absence of IL-12 and IL-18. NK1.1+TCR{beta}- NK cells were gated and intracellular accumulation of IFN-{gamma} was analyzed. Quadrant settings were determined by staining with isotype control mAbs. The percentages of intracellular IFN-{gamma}+ and IFN-{gamma}- NK cell subpopulations are shown. Representative profiles from two independent experiments are shown.

 
Enforced expression of Bcl-2 does not restore the number of NKT cells in IL-2/IL-15R{beta}-/- mice

NKT cells are defined as T cells expressing the common NK cell marker NK1.1 and skewed TCR composed of invariant V{alpha}14-J{alpha}281 chain paired preferentially with polyclonal V{beta}8.2 chain, which recognize CD1d-bound glycolipid ligands (77). NKT cells secrete immunoregulatory cytokines including IL-4 rapidly upon activation (77). In contrast to NK cells, the number of NK1.1+TCR{beta}+ NKT cells in the liver and thymus did not increase significantly in bcl-2-IL-2/IL-15R{beta}-/- mice (Figs. 1Go and 6Go). Because a small number of NKT cells were present in IL-2/IL-15R{beta}-/- and bcl-2-IL-2/IL-15R{beta}-/- mice (Figs. 1Go and 6Go), and V{alpha}14-J{alpha}281 mRNA could be detected in both mice (data not shown), we examined the function of residual NKT cells in these mice. As shown in Fig. 7Go, NKT cells in IL-2/IL-15R{beta}-/- and bcl-2-IL-2/IL-15R{beta}-/- mice could generate a rapid, though diminished, IL-4 response after in vivo stimulation with their specific ligand {alpha}GalCer (78). Fewer NKT cells were present in the spleen cells prepared from IL-2/IL-15R{beta}-/- mice injected with {alpha}GalCer than in those from +/+ mice, but the proportion of IL-4-producing cells in the NKT cell population of IL-2/IL-15R{beta}-/- mice was comparable to that of +/+ mice (Fig. 8Go). In addition, NKT cells were the primary population that produced IL-4 after in vivo stimulation with {alpha}GalCer in both +/+ and IL-2/IL-15R{beta}-/- mice (data not shown). Therefore, the diminished IL-4 production in IL-2/IL-15R{beta}-/- and bcl-2-IL-2/IL-15R{beta}-/- mice in response to {alpha}GalCer stimulation in vivo would be attributed mainly to the reduced number of NKT cells in these mice.



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  		FIGURE 6. Enforced expression of Bcl-2 fails to restore the number of NKT cells in IL-2/IL-15R{beta}-/- mice. Absolute numbers of NKT cells in the liver and thymus of the mice with indicated genotypes were calculated from the total cell number and the percentage of NKT cells. The mean and SD of three mice are shown. *, Significant decreases in numbers of NKT cells as compared with those in the relevant (bcl-2+ or bcl-2-) IL-2/IL-15R{beta}+/+ control mice (p < 0.05).

 


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  		FIGURE 7. Residual NKT cells in IL-2/IL-15R{beta}-/- and bcl-2-IL-2/IL-15R{beta}-/- mice can secrete IL-4 rapidly upon in vivo stimulation with {alpha}GalCer. IL-4 secretion by the spleen cells from {alpha}GalCer-treated (+) and untreated control (-) mice was determined ex vivo. Each bar represents the mean and SD of triplicate cultures. Representative data from two independent experiments are shown.

 


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  		FIGURE 8. Residual NKT cells in IL-2/IL-15R{beta}-/- mice can produce IL-4 in response to in vivo stimulation with {alpha}GalCer. Upper panels, Spleen cells from {alpha}GalCer-treated +/+ and bcl-2-IL-2/IL-15R{beta}-/- mice were stained with anti-TCR{beta} and anti-NK1.1 mAbs. Lower panels, NK1.1+TCR{beta}+ cells NKT cells were gated and intracellular accumulation of IL-4 was analyzed. Quadrant settings were determined by staining with isotype control mAbs. The percentages of NKT cells (upper panels) and intracellular IL-4+ and IL-4- NKT cell subpopulations (lower panels) are shown. Representative profiles from two independent experiments are shown.

 
Enforced expression of Bcl-2 does not restore the number of intestinal IEL in IL-2/IL-15R{beta}-/- mice

Intestinal IEL are divided into several subsets based on the expression of TCR{alpha}{beta}/{gamma}{delta} and CD4/CD8{alpha}/CD8{beta} (79). IL-2/IL-15R{beta}-/- mice display selective reduction in the numbers of the TCR{gamma}{delta} and TCR{alpha}{beta}CD8{alpha}{alpha} subsets of intestinal IEL (23, 25), which are believed to develop extrathymically (79). In bcl-2-IL-2/IL-15R{beta}-/- mice, the relative proportion of the TCR{gamma}{delta} and TCR{alpha}{beta}CD8{alpha}{alpha} subsets of intestinal IEL appeared to increase slightly as compared with those in IL-2/IL-15R{beta}-/- mice (Fig. 9Go). However, the increases in absolute numbers of these subsets were not statistically significant (Fig. 10Go), because of the bcl-2 transgene-induced reduction of the total number of intestinal IEL (Fig. 9Go), which was due to decreases of the TCR{gamma}{delta} and TCR{alpha}{beta}CD8{alpha}{beta} subsets (Figs. 9Go and 10Go). The mechanism of the reduction of intestinal IEL number in bcl-2-IL-2/IL-15R{beta}+/+ mice is currently unknown. Interestingly, MHC class II expression by villus intestinal epithelial cells, which is regulated by the TCR{gamma}{delta} subset of intestinal IEL (80), was not down-regulated in IL-2/IL-15R{beta}-/- mice (Fig. 11Go).



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  		FIGURE 9. Enforced expression of Bcl-2 does not restore the numbers of the TCR{gamma}{delta} and TCR{alpha}{beta}CD8{alpha}{alpha} subsets of intestinal IEL in IL-2/IL-15R{beta}-/- mice. Intestinal IEL were stained with anti-TCR{beta} and anti-TCR{delta} (upper panels) or anti-TCR{beta}, anti-CD8{alpha}, and anti-CD8{beta} (lower panels) mAbs. Lower panels, TCR{beta}+ cells were gated, and their CD8{alpha} and CD8{beta} expression was analyzed. The percentage of each subset is shown. The cell number recovered from each mouse is also shown above the upper panels. Three mice for each type were analyzed independently and representative profiles are shown.

 


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  		FIGURE 10. Enforced expression of Bcl-2 fails to restore the numbers of the TCR{gamma}{delta} and TCR{alpha}{beta}CD8{alpha}{alpha} subsets of intestinal IEL in IL-2/IL-15R{beta}-/- mice. Absolute numbers of the TCR{gamma}{delta} and TCR{alpha}{beta}CD8{alpha}{alpha} subsets of intestinal IEL of the mice with indicated genotypes were calculated from the total cell number and the percentage of each subset. The mean and SD of three mice are shown. *, The number of TCR{gamma}{delta} intestinal IEL in bcl-2-IL-2/IL-15R{beta}+/+ mice was significantly reduced as compared with those in +/+ mice (p < 0.05). **, Significant decreases in numbers of intestinal IEL subsets as compared with those in the relevant (bcl-2+ or bcl-2-) IL-2/IL-15R{beta}+/+ control mice (p < 0.05).

 


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  		FIGURE 11. Normal MHC class II expression by the small intestinal epithelia in IL-2/IL-15R{beta}-/- mice. Sections of the small intestines were stained with anti-MHC class II or isotype control mAb. Positive stains of villus intestinal epithelial cells are obtained in +/+ and IL-2/IL-15R{beta}-/- mice but not in the intestinal IEL-deficient SCID mice. At least three sections for each type were examined independently, and representative fields are shown. Scale bar = 20 µm.

 
Enforced expression of Bcl-2 does not rescue the development of skin IEL in IL-2/IL-15R{beta}-/- mice

Skin IEL in normal mice originate from early fetal thymocytes and express an invariant TCR composed of V{gamma}3 and V{delta}1 chains (81). In IL-2/IL-15R{beta}-/- mice, fetal thymic V{gamma}3+ precursors develop almost normally, but V{gamma}3+ cells are completely absent in the adult skin (27). As shown in Fig. 12Go, V{gamma}3+ skin IEL remained undetectable in adult bcl-2-IL-2/IL-15R{beta}-/- mice.



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  		FIGURE 12. Enforced expression of Bcl-2 does not rescue the development of skin IEL in IL-2/IL-15R{beta}-/- mice. Epidermal cells were stained with anti-TCR{beta} and anti-V{gamma}3 mAbs. The percentage of each subset is shown. The cell number recovered from the ears of each mouse is also shown above each panel. Three mice for each type were analyzed independently and representative profiles are shown.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The importance of IL-15/IL-15R in the NK cell development has been documented in previous studies (25, 28, 29, 30, 31, 82, 83, 84, 85). In this study, we show that enforced expression of Bcl-2 restores the number of NK cells in IL-2/IL-15R{beta}-/- mice. Because endogenous Bcl-2 expression by the residual NK cells in IL-2/IL-15R{beta}-/- mice is not significantly diminished (our unpublished data), the increase in the number of NK cells in bcl-2-IL-2/IL-15R{beta}-/- mice would not result from the accumulation of "leaky" NK cells through Bcl-2-mediated prolonged survival. Therefore, an important role of IL-15R in the NK cell development may be to provide survival signals in their precursors at certain critical developmental stages. Although cytokines other than IL-2/IL-15 also induce nonredundant survival signals in NK cell precursors in vitro (84), these cytokines and IL-15 may be involved in different stages of the NK cell development (84, 86).

Because the {gamma}c is shared by other cytokine receptors, IL-15 may activate specific signals promoting the survival of NK cell precursors through IL-2/IL-15R{beta}. Discrete domains of the IL-2/IL-15R{beta} are known to be involved in inducing expression of the antiapoptotic proteins Bcl-2 and Bcl-xL (59, 60, 61, 62, 63, 64, 65). The cytoplasmic tail of the IL-2/IL-15R{beta} chain is divided into three functional domains (87). The membrane-proximal domain containing the serine-rich region (S-region) recruits Jak1 and is essential to invoke downstream signals (8, 88, 89). The intermediate domain containing the acidic region (A-region) interacts with Src family protein tyrosine kinases and an adapter molecule Shc, and activates Ras/mitogen-activated protein kinase and phosphatidylinositide 3-kinase/Akt signaling pathways (36, 62, 90). The C-terminal half region (H-region) is essential for the recruitment and activation of Stat5 (90, 91). Analysis of the IL-2/IL-15R{beta}-/- mice reconstituted with a mutant form of IL-2/IL-15R{beta} lacking either the cytoplasmic A- or H-region has revealed the importance of the H-region in the NK cell development (92). In contrast, the A-region is dispensable for NK cell development (92), although A-region-dependent activation of Ras/mitogen-activated protein kinase and phosphatidylinositide 3-kinase/Akt signaling pathways also induces Bcl-2 and Bcl-xL expression (59, 61, 62, 63, 64). Therefore, among multiple signaling pathways from IL-2/IL-15R{beta}, which lead to induction of Bcl-2 and Bcl-xL, the H-region-dependent Stat5 pathway (60, 65) may provide nonredundant signals for the survival of NK cell precursors. A crucial role for the Stat5 signaling pathway in NK cell development is further supported by the absence of NK cells in Stat5-/- mice (93, 94).

It was reported that enforced expression of Bcl-2 cannot rescue the NK cell development in {gamma}c-deficient mice (51). Therefore, {gamma}c-dependent cytokine receptors other than IL-2/IL-15R may also play a role in the NK cell development by providing critical signals distinct from survival signals. Previous in vitro studies indicate that these signals could be mediated by IL-7R (86, 95), although IL-7-/- and IL-7R{alpha}-/- mice display normal NK cell development (18, 21, 22). It is possible that the IL-7R-mediated signals required for NK cell development might be compensated for by additional signals from IL-2/IL-15R in vivo. Alternatively, a {gamma}c-dependent cytokine receptor other than IL-7R or IL-2/IL-15R might be involved in NK cell development. Although newly identified IL-21R is a likely candidate for such a receptor (10, 11, 96), IL-21R-/- mice have recently been shown to display normal NK cell development (97).

Interestingly, the rescued NK cells in bcl-2-IL-2/IL-15R{beta}-/- mice do not have cytotoxic activity. Similar developmental arrest of NK cells at a noncytotoxic state and restoration of their cytotoxic activity with exogenous IL-15 have been demonstrated in the bone marrow-ablated mice (83). Therefore, IL-15R-mediated signals are crucial not only for the survival of NK cell precursors but also for the acquisition of cytotoxic activity by NK cells. Because noncytotoxic NK cells in the bone marrow-ablated mice express relatively low levels of Ly-49 receptors (83), IL-15R-mediated signals might be required for the expression of activating Ly-49 receptors by NK cells to recognize target cells. However, we have confirmed that comparable levels of inhibitory and activating Ly-49 receptors are expressed by the noncytotoxic NK cells in IL-2/IL-15R{beta}-/- and bcl-2-IL-2/IL-15R{beta}-/- mice and by the cytotoxic NK cells in +/+ mice. Alternatively, induction of cytotoxic proteins in NK cells might depend on IL-15R-mediated signals. The latter possibility is supported by the observation that perforin expression is regulated by IL-2/IL-15R{beta}-mediated Stat5 activation (98).

In contrast, the rescued NK cells in bcl-2-IL-2/IL-15R{beta}-/- mice can produce IFN-{gamma}. In a recent study (99), in vivo developmental stages of NK cells have been defined, and expansion and functional maturation (acquisition of cytotoxic activity and the capacity to produce IFN-{gamma}) of NK1.1+Ly-49+DX5+Mac-1low immature NK cells was shown to be accompanied by the up-regulation of Mac-1 expression. Most residual spleen NK cells in IL-2/IL-15R{beta}-/- mice express Mac-1 at high levels, and these phenotypically "mature" NK cells increase significantly by enforced expression of Bcl-2. The "mature" NK cells in bcl-2-IL-2/IL-15R{beta}-/- mice have the capacity to produce IFN-{gamma}, but not cytotoxic activity. Therefore, up-regulation of Mac-1 expression, expansion, and acquisition of the capacity to produce IFN-{gamma} and cytotoxic activity, which occur during the final maturation steps in NK cell development, may be regulated by different signaling pathways. In addition, proliferation of NK cell precursors at this developmental stage may not be mediated directly by signals from IL-15R.

NKT cells and the TCR{gamma}{delta} and TCR{alpha}{beta}CD8{alpha}{alpha} subsets of intestinal IEL are reduced, but not absent, in IL-2/IL-15R{beta}-/- mice (23, 25, 26). IL-2/IL-15R-mediated signals do not appear to be crucial for the functional maturation of the precursors of these cells, because at least some functions of residual NKT cells and TCR{gamma}{delta} intestinal IEL in IL-2/IL-15R{beta}-/- mice are not compromised. Therefore, the precursors of NKT cells and intestinal IEL would be able to differentiate to some extent without IL-2/IL-15R, and IL-2/IL-15R-mediated signals might be important for their survival and/or expansion. Because enforced expression of Bcl-2 does not restore the numbers of NKT cells or intestinal IEL in IL-2/IL-15R{beta}-/- mice, the primary role of IL-2/IL-15R in the development of these cells may be to provide proliferation signals.

The absence of V{gamma}3+ skin IEL in adult IL-2/IL-15R{beta}-/- mice is primarily due to the impaired survival and/or expansion of V{gamma}3+ cells in the fetal skin, rather than the developmental block of their fetal thymic precursors (27). Somewhat surprisingly, in light of these previous observations, enforced expression of Bcl-2 cannot rescue skin IEL in adult IL-2/IL-15R{beta}-/- mice. However, it should be noted that some forms of apoptotic cell death, including that mediated by the Fas-Fas ligand interaction, cannot be prevented by Bcl-2 (100). Therefore, IL-2/IL-15R{beta} might provoke survival signals in skin IEL independently of the induction of Bcl-2-related antiapoptotic proteins. Alternatively, continuous expansion of skin IEL through IL-2/IL-15R-mediated proliferation signals might be required for their maintenance in the skin. Our results are consistent with recent observations that introduction of the bcl-2 transgene fails to rescue skin IEL in V{gamma}3 TCR-transgenic IL-2/IL-15R{beta}-/- mice (101).

In conclusion, this study has revealed an important role for the survival signals from IL-2/IL-15R, which are mediated through the induction of Bcl-2-related antiapoptotic proteins, in the development of NK cells. However, additional signals from IL-2/IL-15R are required for the acquisition of cytotoxic activity by NK cells, expansion of the precursors of NKT cells and intestinal IEL, and maintenance of skin IEL. As was the case for the two distinct signals from IL-7R regulating the development of major lymphocyte lineages, heterogeneous signals from IL-2/IL-15R may play differential roles in the development of each innate lymphocyte lineage.


   Acknowledgments
 
We thank K. Ikuta, K. Akashi, H. Suzuki, T. W. Mak, and H. Kawamura for providing mice and mAbs and for helpful discussions, T. Ohteki for critically reviewing the manuscript, T. Imai for technical assistance, and Kirin Brewery for {alpha}GalCer (KRN7000).


   Footnotes
 
1 This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan (to K.K.). Back

2 Current address: Department of Medicine, Division of Medical Oncology and Transplantation, Duke University Medical Center, Durham, NC 27710. Back

3 Address correspondence and reprint requests to Dr. Kazuhiro Kawai, Department of Dermatology, Niigata University School of Medicine, 1-757 Asahimachi-dori, Niigata 951-8510, Japan. E-mail address: kawai{at}med.niigata-u.ac.jp Back

4 Abbreviations used in this paper: {gamma}c, common cytokine receptor {gamma}-chain; {alpha}GalCer, {alpha}-galactosylceramide; IEL, intraepithelial lymphocyte; Jak, Janus kinase. Back

Received for publication February 5, 2002. Accepted for publication August 6, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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